1. Field of the Invention
This invention pertains to organic complexing agents capable of complexing with lanthanide cations and the formed lanthanide chelates useful as fluorescent compounds in the determination of physiologically active materials.
The sensitivity of the analytical methods based on molecular fluorescence is limited by the background signal due to either Roman scattering or fluorescent impurities in the sample. Both of these interferences are generally short-lived phenomena, i.e. the radiative transition following the excitation of the molecule occurs within a microsecond time span. Thus any compound with a long-lived fluorescence can be effectively determined in the presence of short-lived background if a fluorometer with time resolution is at hand. In this fluorometer the sample is illuminated by an intermittent light source such thay the long-lived fluorescence is measurable during the dark period subsequent to the decay of the short-lived background. This invention relates to the synthesis and use of such compounds with the long-lived fluorescence.
2. Description of the Prior Art
The long-lived fluorescence (0.1-3 ms lifetime) of certain chelates of rare-earth metals has been known for some time. The use of these chelates in fluorometric immunoassay (FIA) with time resolution has been described in German OLS 2,628,158 and U.S. Pat. No. 4,374,120. In these publications the complexing agents are aromatic diketones. In German OLS 2,628,158 the fluorescent chelate is "conjugated", i.e. convalently bound to the antigen or antibody. The main shortcoming in this work is the aqueous instability of the chelates which hinders the use of the method at low concentrations.
In Eur. Pat. Appl. 82850077.7 (publ. no. 64484) another ligand is attached either to the antigen or antibody. This ligand is non-fluorescent and serves only in carrying the lanthanide through the separation step of the antigen-antibody complex. After the separation the lanthanide cation is dissociated at low pH and another, fluorescent diketone chelate is formed and measured in aqueous micellar phase. This method gives a very good sensitivity but suffers from somewhat lengthy procedure.
It would be very advantageous to have fluorescent probes with good aqueous stability which would allow shorter assay procedures and also the use of probes in other methods than immunoassay, e.g. in fluorescence microscopy.